Development and Evaluation of a Real-Time FRET Probe Based Multiplex PCR Assay for the Detection of Prohibited Meat and Bone Meal in Cattle Feed and Feed Ingredients

Abstract

A novel real-time fluorescent multiplex polymerase chain reaction (PCR) assay for detecting and discriminating between bovine, ovine, and caprine contaminates in cattle feed was developed that simultaneously performs quality control monitoring on both the DNA extraction process and the level of PCR inhibition in the final DNA extract in a single PCR run. The assay used a single set of primers and two sets of FRET probes targeting the ruminant-specific mitochondrial cytochrome b gene. An internal control PCR reaction targeting a region of the chloroplast RNA polymerase beta-subunit (rpobeta) gene, which is conserved among plants, was incorporated into the ruminant multiplex PCR reaction in order to both monitor the DNA extraction method and to test for the presence of PCR inhibitors. The detection limit for bovine and ovine contaminates was evaluated over a period of two sets of six trials on 15 different types of cattle feed and feed ingredients spiked with known concentrations of bovine meat and bone meal (BMBM) and lamb meat and bone meal (LMBM). The assay was able to detect 0.05% w/w BMBM contamination and 0.1% w/w LMBM contamination in all samples of cattle feed and feed ingredients tested.